In recent years, the development of genetic engineering techniques has had dramatic implications for the field of crop improvement. Using these techniques, beneficial traits can be introduced into almost any crop, and improved crops can be rapidly obtained. The use of genetic engineering obviates the need for lengthy processes that introduce the desired trait through conventional breeding methods.
Present plant transformation methods generally lead to the random integration of transgenes into a host genome. This random integration is problematic for a variety of reasons, including, for example, potentially variable transgene expression resulting from different integration loci, so-called “position effect,” and the risk of mutating the host genome during integration of the transgene. As a result of these potential problems, a large number of transformation events must be screened and tested in order to obtain a transgenic plant exhibiting the desired level of transgene expression without concomitant abnormalities resulting from an inadvertent sequence interruption at an important locus in the plant's genome. Moreover, if a transgenic plant is to be modified by the subsequent addition of one or more transgenes, random integration of the additional transgene(s) renders the implementation of breeding programs for plants containing these multiple transgenes cumbersome and difficult, especially for elite plant lines.
One approach to targeted transgene integration employs site-specific recombinases. Site-specific recombination systems have been identified in several prokaryotic and lower eukaryotic organisms. Such systems typically comprise one or more proteins that recognize two copies of a specific nucleotide sequence, cleave and ligate those nucleotide sequences, and thereby provide a precise, site-specific exchange of genetic information. Several site-specific recombinases are known in the art. These include, but are not limited to, e.g., the bacteriophage P1 Cre/lox system (Austin et al. (1981) Cell 25: 729-736), the R/RS recombinase system from the pSR1 plasmid of the yeast Zygosaccharomyces rouxii (Araki et al. (1985) J. Mol. Biol. 182: 191-203), the Gin/gix system of phage Mu (Maeser and Kahlmann (1991) Mol. Gen.Genet. 230: 170-176), the FLP/FRT recombinase system from the 2 μm plasmid of the yeast Saccharomyces cerevisiae (Broach et al. (1982) Cell 29: 227-234), and the Int recombinase from bacteriophage Lambda (Landy (1989) Annu. Rev. Biochem. 58: 912-949; Landy (1993) Curr. Opin. Genet. Dev. 3: 699-707; Lorbach et al. (2000) J. Mol. Biol. 296: 1175-1181; and WO 01/16345).